Genome-wide identification and comparative analysis of Dmrt genes in echinoderms

The Dmrt (Doublesex-mab3-related transcription factor) gene family is a class of crucial transcription factors characterized by one or several conserved DM (Doublesex/Mab-3) domains. Dmrt family genes can participate in various physiological developmental processes, especially in sex determination/differentiation. Echinoderms are extremely important research objects in various fields, such as sex determination/differentiation and neuroscience. However, to date, the genome-wide characterization and analysis of Dmrt genes in echinoderms have not been investigated. In this study, the identification and analysis of Dmrt genes in 11 representative echinoderms were performed using bioinformatics methods. A total of 43 Dmrt genes have been found in the studied echinoderms, and the number of Dmrt genes in different species ranges from 2 to 5. The phylogenetic tree showed that all Dmrt genes from echinoderms can be subdivided into 5 classes, the Dmrt2-like class, Dmrt3-like class, Dmrt4/5-like class, Dsx-like class, and a novel Dmrt (starfish-specific) class. Furthermore, selective pressure assessment suggested that the Dmrt genes underwent purifying selection pressure. In general, this study provides a molecular basis for echinoderm Dmrt genes and may serve as a reference for in-depth phylogenomics.

The Dmrt (Doublesex-mab3-related transcription factor) gene, including one or several DM (Doublesex/Mab-3) domains, has been widely studied due to various functions [1][2][3][4] , especially in sex determination/differentiation. For example, the Z-linked Dmrt1 gene is vital for sex determination/differentiation in birds 5 . In Xenopus laevis, a W-linked Dmrt gene can be involved in the development of the primary ovary 6 . A Y-specific Dmrt gene, DMY/ Dmrt1bY, can determine the sex of Oryzias latipes [7][8][9] . In general, the Dmrt gene family is an important gene family involved in sex-related development during organism evolution.
To date, Dmrt family genes have been studied in mammals, teleosts, and insects. The members of the Dmrt gene family showed substantial differences in different organisms. For example, eight Dmrt members have been found in some mammals 4 , such as Homo sapiens and Mus musculus. In teleosts, a total of seven Dmrt members have been identified from Larimichthys crocea 10 , and five Dmrt genes have been found in Oreochromis niloticus 11 . In Drosophila melanogaster, only four Dmrt genes have been identified. However, to date, no genome-wide study has been conducted to identify Dmrt genes in echinoderms.
Echinoderms have usually been considered to be the closest invertebrate sister group of vertebrates 12 , with particular evolutionary classification and phylogeny. Meanwhile, as an ancient invertebrate group, echinoderms have diverse reproduction modes, including asexual multiplication, parthenogenesis, hermaphroditism, and dioecy 13 . Therefore, echinoderms are extremely important research objects in many fields, such as sex determination/differentiation 13 , neuroscience 14 , and regeneration biology 15 . In particular, recent studies have suggested that some biological processes in echinoderms are associated with Dmrt genes 16,17 . However, no research has focused on the systematic investigation of Dmrt family genes in echinoderms.
The main research objective of this study was to systematically analyze the abundance of Dmrt genes in echinoderms. With the decoding of many echinoderm genomes, including Acanthaster planci 18 26 , genome-wide identification and analysis of the Dmrt gene family was carried out. Furthermore, the functional domains and sequence structures of the Dmrt genes were predicted, Sequence analyses and genomic distribution of Dmrts. The general feature format file was used to reveal the Dmrt gene structure and exon information. The conserved motifs of the Dmrt genes were predicted by using MEME 33 with the following options: largest number, 25; minimum length, 6; maximum length, 50; and default values for other parameters. Conserved motifs and gene structure were both visualized by TBtools v1.098 29 . In addition, the conserved domains of all identified DMRT genes were analyzed using the Batch SMART plug-in in TBtools software (version 1.098) 29 and visualized with the iTOL (interactive tree of life) online tool 32 . The genomic distribution was visualized with gene arrow maps generated by using the gggenes package in R 34 .
Selective pressure assessment. Selective pressure was assessed by using the branch and site model in EasyCodeML V1.0 35 . The branch models assume that the ratios (ω) of nonsynonymous substitution sites (dN) and synonymous substitution sites (dS) vary among branches. Under the branch model, the comparison of two models (one ratio and free ratio) was calculated to test whether ω is different among different branches. The site models assume that the ω ratio varies among sites. Under the site models, the specific models (M0, M1a, M2a, M3, M7, and M8) were tested by adjusting the parameters. Among these models, comparison of M3/M0 is used to detect whether the ω ratio between different sites is consistent, while the comparisons of the M2a/M1a and M8/M7 model pairs test for positive selection.

Results
Identification and characterization of Dmrt genes. A total of 43 Dmrt genes have been identified in 11 representative echinoderms. The amino acid sequences of the identified Dmrt genes are provided in Supplementary Table S3. The number of Dmrt genes in each species ranges from 2 to 5, which are listed in Table 1. The characteristics of all the identified proteins in echinoderms were predicted and are listed in Table 1. The results showed that the biophysical properties of different Dmrt proteins were different. AA length varied from 110 to 794. The MW ranged from 12,660.77 to 88,920.97 Da, while the PI values varied from 5.44 to 10.37. Additionally, the vast majority of Dmrt proteins were considered unstable (instability index greater than 40).

Phylogenetic analysis of Dmrt genes.
To understand the evolutionary relationships of Dmrt genes in echinoderms, a phylogenetic analysis was carried out using Dmrt protein sequences from vertebrates and invertebrates. As shown in Fig. 1, 43 Dmrt genes from echinoderms were divided into 5 classes: the Dmrt2-like class, Dmrt3-like class, Dmrt4/5-like class, Dsx-like class, and novel Dmrt class. The Dmrt2-like class contains 11 genes from 11 echinoderms. The Dmrt3-like class consists of 11 Dmrt genes from 10 echinoderms. Nine Dmrt genes from 8 echinoderms have formed a Dmrt4/5-like class. The Dmrt genes in the Dsx-like class come from sea urchins, sea cucumbers, and crinoids. The remaining genes from starfish were divided into a novel Dmrt class.

Sequence analyses and genomic distribution. The exon-intron diversification among echinoderm
Dmrt genes is also displayed in Fig. 2. The exon numbers of Dmrt genes in 11 echinoderms varied from 1 to 6. Genes in the same class have more similar exon-intron structures. In addition, although all the predicted Dmrt proteins contain motif 1, the proteins in the same class have more similar motif structural features. By using the Batch SMART search, it was found that all Dmrt genes include a DM domain, and some Dmrt genes contain Pfam:DMA (Fig. 3). The genomic locations of Dmrt genes in different species are shown in Fig. 4

Discussion
The Dmrt gene family has been identified genome-wide in various animal groups, including mammals, insects, and teleosts 10,36,37 . However, little is known about aquatic invertebrates. In particular, a comprehensive survey of Dmrt genes has not been carried out in echinoderms, although some echinoderm genome sequences have been available for several years. In the current study, a systematic analysis of Dmrt family genes was performed in 11 echinoderm genomes. Two to five Dmrt genes have been identified in different echinoderms. According to previous studies, the difference in the number of Dmrt genes may be related to genome size and genomic duplication rounds 37 . Although Dmrt genes are widely represented across the animal kingdom, they present a certain degree of species specificity. For example, Dmrt1 is found only in vertebrates, and Dmrt 6-8 is only present in mammals. This pattern was confirmed in this study. No Dmrt1-like gene or Dmrt6/7/8-like gene was identified in echinoderms, while Dmrt2-like, Dmrt3-like, Dmrt4/5-like and possibly Dsx-like genes were found in this study. Similar Dmrt members can also be found in Panarthropoda 38 . These results imply that Dmrt2, Dmrt3, Dmrt4/5, and Dsx-like genes may be widely present in invertebrates. Unlike some fish that harbored two paralogs of Dmrt2 (Dmrt2a and Dmrt2b), all the studied echinoderms carried one Dmrt2-like gene, suggesting that the Dmrt2-like gene may be conserved in echinoderms. Dmrt2 has very important roles in sex reversal, testicular development, and embryonic development. For example, in humans, Dmrt2 is associated with XY sex reversal and gonadal dysgenesis 39 . Dmrt11E was proven to be a crucial factor for gametic formation in domesticated silkworm 40 . Analogous functions of Dmrt2 have also been reported in several aquatic invertebrates. In Penaeus monodon, Dmrt11E was proposed to affect muscle development, testis development, spermatogenesis, and somites 41 . In Chlamys nobilis, Dmrt2 is expressed exclusively in gonads, implying that it may be involved in the maintenance of gonadal function or gonadal development 42 . In addition, in zebrafish, Dmrt2 was found to have a function in regulating the left-right patterning of the mesoderm 43 . In summary, given that Dmrt2 has diverse functions, Dmrt2-like genes in echinoderms should be further studied.
In this study, both Dmrt2-like genes and Dmrt3-like genes were found in all species except P. borealis. Furthermore, the Dmrt2-like/Dmrt3-like cluster can be found in numerous echinoderms. These results may support the previous conclusion that Dmrt3 may have emerged through a gene duplication event of Dmrt2 during deuterostome evolution 44 . In addition, functional investigations on Dmrt3 have only been performed in vertebrates, showing that this gene can play pivotal roles in configuring the spinal circuits controlling stride 45 . Consistent with previous findings, the current phylogenetic analysis showed that Dmrt4 and Dmrt5 were clustered into a major branch, suggesting that these two types of genes originated from the same ancestor of Dmrt. In this study, the Dmrt4/5-like gene was identified in 8 echinoderms with the exception of H. pulcherrimus, T. reevesii, and P. borealis. In particular, the Dmrt4/5-like gene was duplicated in P. miniata. To date, Dmrt4 and Dmrt5 have been found to be closely related to neurogenesis. For instance, in Xenopus, Dmrt4 and Dmrt5 are important regulators of olfactory placode neurogenesis 46 . During the development of the hippocampus in mice, Dmrt5 was shown to be involved in the regulation of the neuron-glia cell-fate switch 47 . A similar function was also observed in invertebrates. In Drosophila, Dmrt99B plays an essential role in initiating temporal patterning in medulla neuroblasts. Thus, it will be interesting to investigate whether Dmrt4/5 play similar functions in echinoderms. www.nature.com/scientificreports/ Dsx was found to have a pivotal role in sexual dimorphism in genetic sex-determining animals, including insects and nematodes. In Drosophila, Dsx has male-and female-specific isoforms (DsxM and DsxF), which can regulate different target genes, resulting in sex-specific morphology 48 . In B. mori, two Dsx isoforms (BmDsxF and BmDsxM) can enhance male and female differentiation in gonads and external genitalia, respectively 49 . DapmaDsx1 (Dsx ortholog) in Daphnia magna was confirmed as a critical regulator of the male phenotype 50 . In this study, a possible Dsx-like gene class was found in the phylogenetic tree. However, whether these genes have similar functions is unclear. In addition, it should be noted that the Dsx gene class in the phylogenetic tree was backed by low bootstrap values. In particular, these sequences from sea urchins, sea cucumbers, crinoids, and two Dsx sequences from shrimp seem to be unrelated to the Dsx from A. aegypti, B. mori, and D. melanogaster. This result may be caused by relatively few informative characters outside of the DM domains. In particular, it www.nature.com/scientificreports/ is worth noting that the members in the Dsx-like cluster present different protein characteristics. For example, the AA and MW of the Dsx-like gene from sea urchins were significantly higher than those of the Dsx-like gene from other species. These results may imply that the function of Dsx-like genes in sea urchins may be different from that of Dsx-like genes in other species. Moreover, a starfish-specific Dmrt class was identified in the current study. These genes were phylogenetically distant from the other Dmrt members. Their exon-intron structure is also unique. Similar results can be learned in other aquatic invertebrates. Comparative phylotranscriptomics revealed that DMRT1L is a mollusk-specific gene 51 , and a novel Dmrt gene (EsDmrt-like) was identified in Eriocheir sinensis 52 . These results indicate that there may be more members of the Dmrt gene family, especially in aquatic invertebrates. Therefore, it is necessary to conduct systematic identification and analysis of the Dmrt gene family in other classes of invertebrates.

Conclusion
In this study, a systematic analysis of Dmrt family genes in 11 representative echinoderms was performed. A total of 43 Dmrt genes have been found, and the number of Dmrt genes in different echinoderms ranges from 2 to 5. The phylogenetic tree showed that all Dmrts from echinoderms were classified into 5 classes: the Dmrt2-like class, Dmrt3-like class, Dmrt4/5-like class, Dsx-like class, and novel Dmrt class. Furthermore, selective pressure assessment suggested that the Dmrt genes underwent purifying selection pressure. In general, this study provides a molecular basis for echinoderm Dmrt and may serve as a reference for in-depth phylogenomics.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.